BRD4-mediated epigenetic regulation of endoplasmic reticulum-mitochondria contact sites is governed by the mitochondrial complex III

This is a high content drug screening dataset composed of images and single-cell analysis results from a 384-well plate The SPLICS reporter is composed of a split GFP1-10 barrel localized to the mitochondrial outer membrane, a P2A self-cleavage signal for equimolar expression, and the remaining beta11 fragment targeted to the ER membrane. Upon ERMCS formation within the 8-10 nm distance, the split-GFP reporter forms intact GFP. Doxycycline-inducible SPLICS reporter stable cell lines were generated via a three-plasmid PiggyBac transposase system. Cells were co-transfected with the plasmids using Lipofectamine 2000 transfection reagent, then the next day selected with 2 mg/ml of G418 (Geneticin) for 7 days. With the SPLICS integrated cells, mitochondrial TagRFP is expressed in them. For mitochondrial TagRFP labeling in cells, we utilized a lentiviral backbone and introduced a mitochondria matrix TagRFP (mitoTagRFP) to control for gene expression and monitor mitochondrial abundance. After mitoTagRFP integration, 100 ng/ml of doxycycline were used to induce SPLICS reporter expression, and GFP/TagRFP double positive cells were sorted on Bigfoot Spectral Cell Sorter (Invitrogen) to isolate GFP/TagRFP positive clones. Clones were cultured and screened for accurate localization of SPLICS reporter to mitochondria and ER via live cell imaging and for normal mitochondrial oxygen consumption rate. Probe induction did not have a deleterious effect on cell number or increasing green fluorescence for at least 72 hours of reporter induction. For ERMCS analysis with SPLICS, cells will be induced with 100 ng/ml of doxycycline for minimally 24 hr before treatment or analysis.